See also DNA samples; High purity samples; Hygroscopic samples; Low ionic strength samples; Magnetic samples; Semisolid samples; Viscous samples as affecting balance accuracy, 55 with pH
Trang 1Reference electrodes
with pH meters, 77–78
in pH meters, 80
Refillable electrodes
in pH meters, 87, 91–92, 93
storage of, 91–92
Refrigerated centrifuges,
troubleshooting, 66
Refrigerators, storing reagents in,
41– 42
Refrigerator shelves, storing reagents
on, 42
Regeneration, of oligo(dT)-cellulose,
211–212
Regulating eukaryotic expression,
509–510
Reimbursement, for company
liability, 28
Relationships, with sales
representatives, 16–17
Reliability
of balances and scales, 54–55
of data, 6–7
of products, 14
of reagents, 39–40
of suppliers, 13
Remeta, David, 267
Replication, data reliability and, 6
Reporting, of research results, 8–9
Reprobing
in hybridization, 432–433, 433–434
in Western blotting, 379, 380, 388–389
Reproducibility
of DNA purification methods, 169
in electrophoresis, 341–343
improving in electrophoresis, 353
with polymerase chain reactions, 298
of storage phosphor images, 443
Reproduction, of research results, 8
Research
defending, 8–9
motivation for doing, 8, 9
reporting of, 8–9
sales representatives in, 15–16
successful, 5–9
Research planning, 3
reporting results and, 8
Research style, 2
Resins, in RNA purification, 210
Resolution
of autoradiography film, 438
improving in electrophoresis, 353
of labels, 405–406
of pH meters, 85
of spectrophotometers, 97–98
of storage phosphor imagers, 441–442, 446
Resources, in project planning, 2–3 Response time, of pH meters, 93 Restriction endonucleases properties of, 232–236 quality control assays for, 234–235 star activity of, 229–230
Restriction enzymes, 226–262 altering specificity of, 248–250 commercially available, 226–227 complex digests with, 239–244 costs of, 227–228
double digests with, 242–244 easily used, 229
genomic digests with, 244–255 methylation sensitivity of, 231 quality control data for, 233–235 restriction endonucleases as, 232–235
selecting, 229–231 shipments of, 259–260 simple digests with, 236–239 site preference of, 230 stability of, 236 titer assays for, 255–259 transformation failures with, 260–262 troubleshooting, 255–262
Restriction length fragment polymorphism (RFLP) analysis, 226
Restriction sites, creating rare or unique, 247–255
Reuse, of oligo(dT)-cellulose, 211–212 Reverse dot blots, in RNA
purification, 200 Reverse osmosis, water purification via, 43– 44
Review, data gathering and, 5 Ribogreen dye, quantitating dilute RNA via, 219
Ribonuclease protection assays, in RNA purification, 201, 203 Ribonucleotides
nomenclature of, 269 quantitating solutions of, 273–275 Riis, Peter, 373
Ring badges, monitoring radiation exposure with, 161
Trang 2RNA See also Nucleotides;
Oligonucleotides;
Polynucleotides centrifugation of, 63
as hybridization target, 402 solutions of polymers of, 287–288 storage of, 214–215, 219, 222 RNA purification, 198–222 integrity of RNA from, 202–203 lysis in, 215
maximizing yield from, 212–219 pauses during, 218
predicting yield from, 201–202 storing RNA from, 219 strategies for, 198–212 troubleshooting, 220–222 RNA sample preparation, for polymerase chain reactions, 312 RNase-free techniques, 212–214 RNase inhibition, 215
RNases DNA contamination with, 169
in DNA extraction, 173 RNA contamination with, 212–213
in RNA degradation, 214, 215, 217, 219
Robinson, Derek, 225 Room temperature storage, of reagents, 40
Rotor identification codes, for centrifuges, 62
Rotors, for centrifuges, 57, 58–61, 62–63, 64
RT-PCR technique, 314
in RNA purification, 200, 201, 202, 203
troubleshooting, 221–222, 321–322 RTPs, nomenclature of, 269
Saccharomyces cerevisiae
as biohazard, 115, 128 recognition sites in, 248
S-adenosylmethionine, in simple
digests, 239 Safety
with acrylamide, 334–336 with biological materials, 114–140 electrical, 336–337
with radioactive materials, 133, 142–165, 166
Safety equipment, for biosafety, 119 Safety glasses, 118–119
Sales representatives, 14–18 expectations of, 15–16 functions of, 15 leverage via, 17–18 motivations of, 16 for pH meters, 94 relating to, 16–17
Salmonella typhi, as biohazard, 114,
115
Salmonella typhimurium, recognition
sites in, 248 Salt pellets, in RNA purification, 205–206
Salts
as buffers, 33, 35
in DNA precipitation, 174–175, 185, 189
in hybridization buffers, 427, 428–429
for sequential double digests, 243 for Western blotting buffers, 382 Sample collection, minimizing RNA degradation during, 214–215 Sample concentration
absorbance and, 104–105, 108–109
in spectrophotometry, 103 Sample disruption, minimizing RNA degradation during, 215–218
Sample handling See also DNA
samples; High purity samples; Hygroscopic samples; Low ionic strength samples; Magnetic samples; Semisolid samples; Viscous samples
as affecting balance accuracy, 55 with pH meters, 93–94
in Western blotting, 382–383 Sample location, as affecting balance accuracy, 55
Sample matrix, for pH meters, 85–86 Sample preparation, for polymerase chain reactions, 311–312 Sample volumes
of cuvettes, 100 for pH meters, 86 for spectrophotometers, 95 Scales, 51–55
calibrating, 55 Scaling up
of eukaryotic expression, 514–515, 527
of gene expression, 478–479
Trang 3adhering to, 7
in project planning, 4
Schlieren, in acrylamide
polymerization, 342
Science, motivation for doing, 9
Scientific literature, 5–6
Scientists, companies and, 12
Scientist Web site, 14
Scintillation counters, 155
Screens, for storage phosphor
imagers, 444, 445–447
SDS-PAGE, 337, 338, 349
electrical power for, 350–353
native PAGE versus, 345–348
problems with, 480–481
standardized gels for, 363
Sealed containment rooms, in
biosafety, 117
Secondary antibodies, 384
problems with, 395
Secondary decomposition, of
radioisotopes, 156
Secondary reagents
problems with, 395
species specificity in, 385
in Western blotting, 384–387
Secondary structures, in gene
expression, 467
Secreted proteins, expressing, 527–528
Sedimentation coefficient, 55–56
Self-decontamination, 132
Self-inoculation, accidental, 123
Self-monitoring, in radioactive work
areas, 161
Semimicro balances, 51
Semisolid samples, measuring pH of,
90
Sensing electrodes, in pH meters, 77,
80
Sensitivity
of autoradiography film, 438
of direct versus indirect labeling,
410
of hybridization experiments, 403
with polymerase chain reactions,
299–300
of storage phosphor imagers,
441–442, 445
in Western blotting, 377
Sequence information, for eukaryotic
expression, 499
Sequencing cells, constant power electrophoresis with, 352–353 Sequencing gels, constant power electrophoresis with, 352 Sequential double digests, 243–244 Serial numbers, for products, 25 Serum
as blocking agent, 381
in eukaryotic expression, 511–512 Service calls
for balances, 55 for centrifuges, 64–66 for pH meters, 94 for spectrophotometers, 106 Service engineers, for pH meters, 94 Sharps, proper disposal of, 120 Shatzman, Allan R., 491 Shearing, in DNA purification, 169 Shelf life, 22
of acrylamide, 336
of hybridization buffers, 431–432
of labeled probes, 411 maximizing purified DNA, 172
of membranes after crosslinking, 423–424
of nucleotides, 270–271
of oligonucleotides, 280
of radioisotopes, 149–150, 156–
157
of reagents, 40
of restriction endonucleases, 232
of restriction enzymes, 228 Shelves, storing reagents on, 40, 42 Shielding, in minimizing radiation exposure, 163
Shigella flexneri, as biohazard, 115
Shipments, of restriction
endonucleases, 232, 259–260 See also Radioactive shipments
Short term monitoring, in radioactive work areas, 161
Shower, for biosafety, 119 Side-by-side comparisons, 26 Signal duration
in hybridization experiments, 407
in Western blotting, 377 Signal sequences, in protein expression, 468–469 Signal smears
in electrophoresis, 356–357
in genomic digests, 244 nuclease contamination and, 169
Trang 4in polymerase chain reactions, 317–318
troubleshooting, 222 Signal strength, of labels, 405–406 Silica resins, in DNA extraction, 176–178, 186, 190
Silver/silver chloride reference system for pH meters, 77
Simple digests, 236–239 modifying reaction conditions in, 237–239
Simple two-fold titration, troubleshooting with, 255–257 Simultaneous double digests, 242–243 Single-beam spectrophotometers, 95 Single-coated autoradiography film, 438
Single nucleotide polymorphisms (SNPs), polymerase chain reactions for finding, 313 Single-stranded DNA, A260values for, 278
Single-stranded markers, in electrophoresis, 356, 364 Single-stranded nucleic acid polymers nomenclature of, 281–282
solutions of, 288 Single-vector systems, for eukaryotic expression, 510
Site preference, of restriction enzymes, 230
Six problem-solving steps, 20–23 example of, 23–25
Skin keratin, electrophoresis band from, 368
Slides, preparation of, 121 Small companies, 12–13 Smeared signals
in electrophoresis, 356–357
in genomic digests, 244 nuclease contamination and, 169
in polymerase chain reactions, 317–318
troubleshooting, 222 Smith, Tiffany J., 197 Sodium phosphate buffers, in DNA purification, 179
Software for BLAST searches, 328 for selecting primers, 327 for storage phosphor imagers, 444 Solubility, of proteins, 481–483
Solubility problems, in baculovirus experiment, 532
Solution nucleotides purity of, 269–270 stability of, 270–271 Solutions
quantitating dilute RNA, 218–219 RNase-free, 213
Solvents radioisotopes in, 146–147 water as, 44
Sonication, in DNA extraction, 173 Southern blotting, 244, 449–453 Species specificity, in secondary reagents, 385
Specific activity, of radioisotopes,
145–146, 153–154 See also
Radioactivity Specificity, with polymerase chain reactions, 297
Spectinomycin, in plasmid purification, 182 Spectral bandwidth resolution, of spectrophotometers, 97–98 Spectrophotometers, 94–110 accuracy of, 96–97, 101–103, 103–104, 104–105, 105–106 calibrating, 96–97, 98–100 computers with, 95 cuvettes for, 100 light sources for, 95–96 limitations of, 102–103 maintenance of, 101, 107 operating, 107–110 resolution of, 97–98 selecting, 94–98 service calls for, 106 types of, 95–96 wavelength range of, 96 Spectrophotometry quantitating dilute RNA via, 218–219
quantitating nucleotide solutions via, 273, 275
troubleshooting RNA, 221 Spectroscopy, quantitating nucleotide solutions via, 273, 275
Spending limits, 18 Spills
in biosafety, 123
in centrifuges, 66 Spin columns, in RNA purification, 211
Trang 5Spinner flasks, in eukaryotic
expression, 514
Spinning vacuum chambers,
concentrating radioactive
solutions via, 164–165
Spodoptera frugiperda, in eukaryotic
expression, 503, 525
Spore-forming filamentous fungi, safe
handling of, 127–128
Spun column chromatography
through gel filtration resins, in
DNA purification, 178–179,
184–185
Spyro Ruby stain, 359
Stability See also Heat stability
of cell proteins, 464–465
of labeled probes, 411
of nucleotides, 270–271, 275, 276
of oligonucleotides, 280
of radioisotopes, 157–158
of restriction enzymes, 236
of spectrophotometers, 99–100
Stable buffers, 37–38
Stable expression systems, for
eukaryotic expression, 504–506
Stains
high background, 362, 395–396
for nucleic acids (table), 360
for nucleic acid transfer, 419–420
problems with, 395
for proteins (table), 360
selecting for electrophoresis,
357–363
Standard deviation, calculating, 75–76
Standards
in experiments, 21
NIST and, 83–84
for protein quantitation assays,
109–110
for spectrophotometers, 98–100
Standards and guidelines, for pipette
calibration, 70–71
Staphylococcus aureus
as biohazard, 115–116, 131
preparing for pulsed field
electrophoresis, 245–246
Star activity, of restriction
endonucleases, 229–230
Start codons, in gene expression, 466
Statistics, data reliability and, 6
Sterilization, 116–117, 124–125 See
also Autoclaves
by media room, 136
of nitrocellulose and nylon membranes, 417–418
of plastic materials, 135 Stevens, Jane, 49, 77 Stirred tank bioreactors, in eukaryotic expression, 515
Stock solutions, buffers from, 36–37 Storage
of acrylamide, 336
of buffers, 37–38
of hybridization buffers, 431–432
of labeled probes, 411
of membranes after crosslinking, 423–424
of microbial strains, 125–126 minimizing RNA degradation during, 214–215
of nucleic acid polymers, 287–288
of nucleotides, 270–271
of oligonucleotides, 280
of pH meters, 91–92
of pipettes, 68
of purified DNA, 172
of purified RNA, 219, 222
of radioactive materials, 156–159
of reagents, 39, 40– 41, 41– 42
of restriction endonucleases, 232
of Western blots, 383
of Western blotting antibodies, 384 Storage conditions, as source of problems, 21–22, 40– 42 Storage phosphor imagers dynamic range of, 442– 443 erasure of, 447
in nucleic acid hybridization, 441–448
operation of, 441 problems with, 447– 448 quantitative capabilities of, 443– 445 resolution of, 441– 442
screens for, 445– 447 sensitivity of, 441– 442 speed of, 441– 442 Straight percent gels, 345–346 Stray light, as affecting spectrophotometer accuracy, 97, 99
Streaking, 122 Strength, of hybridization
membranes, 414 See also Signal
strength
Trang 6Streptavidin amplification and, 387–388 problems with, 395, 396
in Western blotting, 381–382, 386–387
Streptomyces achromogenes,
restriction enzymes from, 227 Stripping
in hybridization, 432–433
in Western blotting, 379, 380, 383, 388–389
Strong acids, in buffering, 36 Strong bases, in buffering, 36 Structure, of polynucleotides, 283 Subcloning, in eukaryotic expression, 500
Suboptimal growth conditions, in baculovirus experiment, 530–531 Substrates, in complex digests, 239–241 Successful projects, 4
Successful research, 5–9 Sulfur radioisotopes, 157–158 autoradiography film and, 438, 439 shielding for, 163
signal strength of, 406 Supercoiled DNA, centrifugation of, 63–64
Suppliers, 11–29 communicating needs to, 18–19 contacting, 26–29
expectations of, 28 ordering custom products from, 18–19
problems with, 19–29 products from, 13–14 reliability of, 13 sales representatives of, 14–18
in solving problems, 25–26 working with, 12–14 Supplies, for biosafety, 119 Surgical instruments, autoclaving of, 134
Suspensions, proper handling of microbial, 122
Swinging bucket rotor, 58, 59 SYBR Gold dye, 420 quantitating dilute RNA via, 219 SYBR Green dye, 420
quantitating dilute RNA via, 219 Synaptic complex formation, restriction endonucleases and, 254 Synthetic polynucleotides, 281–288
Tags
in eukaryotic expression, 515–517 with fusion systems, 472–474, 474–475 Tap water, 42
organic compounds in, 45–46 Taq DNA polymerase
in DNA purification, 170–171
in polymerase chain reactions, 292 primers with, 313
TaqI methylase, in genomic digests,
250–251 Target pH, buffering toward, 33–34 Targets
for eukaryotic expression, 493–495, 501–502
of hybridization experiments, 402 Task planning, 2
TBE (Tris, borate, EDTA) buffer, in DNA purification, 170
Technical problems, in project planning, 3
Technical support, for pH meters, 94 TEMED potency, in acrylamide polymerization, 342–343 Temperature
in acrylamide polymerization, 343
as affecting balance accuracy, 52–53, 54–55
for autoradiography film detection, 440–441
for baking membranes, 422
in DNA precipitation, 174
in gene expression, 478 for hybridization, 424–425 nucleotides and, 275
pH meters and, 84–85, 86 during pipette testing, 73, 75
in plasmid purification, 180–181 for polymerase chain reactions, 302–303, 309–310
of restriction enzyme shipments, 259–260
storage phosphor imagers and, 446
in storing purified DNA, 172
in storing radioisotopes, 156–157 Temperature compensation, for pH meters, 84–85
Template contamination, minimizing
in polymerase chain reactions, 306–308
Template modification, in polymerase chain reactions, 312
Trang 7Templates, troubleshooting PCR, 315
TenBroeck homogenizer, cell
disruption via, 216
Test liquids, in pipette testing, 73
Test points, in pipette testing, 74
Test volumes, in pipette testing, 74
Therapeutic proteins, eukaryotic
expression of, 493, 494, 496,
501–502
Thermal degradation, of nucleotides,
275, 276
Thermocycling
nucleotide stability and, 275, 276
in polymerase chain reactions,
309–310
The Scientist Web site, 14
Thickness, of hybridization
membranes, 414–415
30-mer oligonucleotide, labeling in
hybridization experiments, 404
Tilt, as affecting balance accuracy, 53–
54
Time
autoclaving, 135, 136
for autoradiography film detection,
440
for complex digests, 242
in DNA precipitation, 174
in DNA purification, 168
for hybridization, 426–427
for media culture requests, 137
in minimizing radiation exposure, 162
for signal detection, 406
for stains, 361
for storage phosphor imager
detection, 441–442
Tip immersion, of pipettes, 69
Tissue
accidental self-inoculation with, 123
isolating DNA from, 172–184
total RNA yield from, 201–202,
203–206, 207–209
Titer assays, troubleshooting with,
255–259
Toluene, radioisotopes in, 147
Top-loader balances, 51
Total RNA, purification of, 198–201,
203–206, 207–209, 212
Toxicity, of proteins, 469–470 See also
Neurotoxin
Transfection, in eukaryotic
expression, 513
Transfer buffers, for nucleic acid transfer, 418–419
Transfer membranes, with Western blotting, 379–380
Transfer vectors, selecting, 524–525 Transformation failures, with restriction enzymes, 260–262 Transient expression systems, in eukaryotic expression, 502–503 Transilluminators, crosslinking on, 422 Translation, with prokaryotic
promoters, 464 Translation termination, problems with, 483–484
Trichoplusia ni, in eukaryotic
expression, 503, 525–526 Trill, John J., 491
Triple helix formation, with Achilles’
heel cleavage, 253 Triple helix resins, in plasmid purification, 183
Tritium autoradiography film and, 438–439
as radioactive waste, 158 shielding for, 163 signal strength of, 406 storage phosphor imagers for, 446
Troubleshooting See Problems
Troutman, Trevor, 49, 51
Trypanosoma, as biohazard, 128
Tubes, for centrifuges, 61 Tungsten lamps, in spectrophotometry, 107, 108 2-D gels, 365–366
pH gradients and, 366–368 2-kilobase DNA fragment, labeling in hybridization experiments, 405 Two-phase extraction systems, for DNA and RNA precipitation, 176
Two-step mRNA (poly(A) RNA) purification, 209
Type A License of Broad Scope, for handling radioisotopes, 143 Tyre, Thomas, 11, 267
Ultracentrifuges, 57 Ultramicrobalances, 51 Ultraviolet-visible (UV/Vis)
spectrophotometry See
Spectrophotometers Unexpected results, planning for, 3
Trang 8Unit definition for restriction enzymes, 233 for storage phosphor imagers, 444 Upper management, directing complaints to, 28–29
UV (ultraviolet) crosslinking, of nucleic acids, 422
UV lamps germicidal, 116 maintaining, 107
in spectrophotometers, 103 Vacuum baking, of membranes, 422 Variables, controlling, 7–8
Vented flammables cabinets, storing reagents in, 40
Vertical rotors, for centrifuges, 63, 65 Vibration, in centrifuges, 66
Viral lytic systems, for eukaryotic expression, 503–504
Viral production problems, in baculovirus experiment, 531 Viruses, safe handling of, 126–127 Virus purification, 171
Viscous samples, measuring pH of, 90 Volatile nuclides, hoods for, 163 Volny, William R., Jr., 141 Volume range
of eukaryotic expression, 514–515
of pipettes, 67–68, 71–72 Volumetric flasks
in eukaryotic expression, 514 refrigerating reagents in, 41 Volumetric titration, troubleshooting with, 257
Walking centrifuge, 66 Walsh, Paul R., 225 Wash buffers, for Western blotting, 382
Washing
in hybridization, 434–435 problems with, 395
in Western blotting, 382–383 Washing efficiency, in hybridization, 435 Wash solutions, in hybridization, 434–435
Waste, disposal of radioactive, 158–159 Waste decontamination, 139–140 Water, 42–47
grades of, 42–44 leaks and, 46–47
microbial contamination of, 45–46 organic compounds in, 45–46
pH of, 44
as solvent, 44 Water purification, via reverse osmosis, 43–44
Water purification systems, leaks in, 46–47
Water purity, in acrylamide polymerization, 344 Wave Bioreactor, in eukaryotic expression, 514–515 Wavelength accuracy, of spectrophotometers, 96, 97, 99, 105 Wavelength range, of
spectrophotometers, 96 Wavelength reproducibility, of spectrophotometers, 99 Weak acids, as buffers, 33 Weak emitters, autoradiography film and, 438–439
Web sites Biosci, 13 for polymerase chain reactions, 328–329
Weighing, quantitating nucleotide solutions via, 274, 287 Western blotting, 374–397 amplification in, 387–388 antibodies in, 378 blocking in, 380–382 detection strategies with, 375–380 gene expression and, 477
molecular weight and, 365 primary antibody in, 383–384 proteins and, 374–375 reprobing in, 379, 380, 388–389 secondary reagents in, 384–387 setting up new methods for, 396–397
stained proteins and, 363 stripping in, 379, 380, 383, 388–389 troubleshooting, 389–396
washing in, 382–383 Western blotting troubleshooting logic tree, 390–392
Wet membranes
in hybridization, 432
in nucleic acid transfer, 420–421 Wick junctions, in pH meters, 93 Wipe test, for radioactive shipments, 151–152
Trang 9World Health Organization (WHO),
115
Xenon lamps, in spectrophotometry,
107
X rays
autoradiography film and, 439–440
in Bremsstrahlung, 152
Yeast
disruption of, 217
eukaryotic expression with,
505–506
minimizing degradation of RNA
from, 215, 217
total RNA isolation from, 208–
209 Yelling, 29 Yields from fusion systems, 471–472 maximizing eukaryotic expression protein, 529–530
Z factor, of pipettes, 75, 76
“Zippering up,” hybridization times and, 426
Zonal centrifugation, 55–56 Zwitterionic detergents, for native PAGE, 355
Zymolase, cell disruption via, 217